1. Field of Invention
This invention relates to a new reagent for use in a new method for the determination of inorganic phosphate in biological fluids, which have or have not been made protein free, by the use of a reducing agent selected from the group consisting of ferric ammonium sulfate (FAS) stannous chloride, and ascorbic acid, in the presence of a lineating surfactant.
2. Prior Art
It is known to the art to reduce molybdenum phosphates to molybdenum blue. Various methods and the reducing agents used therein have been discussed by Henry in Clinical Chemistry, (Harper & row, 1968).
The patent literature also discusses such processes. Thus Daly et al in U.S. Pat. No. 3,795,484 disclose an automated method for the determination of inorganic phosphate in serum. However at column 2 line 19 it specifically states that a protein free serum sample is required for the test.
U.S. Pat. No. 3,547,586 discloses the use of PVP to speedup the formation of yellowish phospho-molybdate prior to the reduction step. Set forth therein also is a synopsis of the Hycel Inc. determination which does not require protein precipitation, but which is slow, 35 minutes, and fails to obey the Beer-Lambert Law. The Hycel procedure also suffers from the fact that glucose levels above 200mg. percent interfere with the test, and since the test is often desired for diabetics, there is limited utility since diabetics are excluded from its use. Note also that the Hycel reactant is stable for about 1 year, provided that it is refrigerated.
A very recent patent is the one to Morin & Prox, 3,853,469 which uses ortho-phenylenediamine or a salt thereof with an acid as the reducing system, with or without a dipolar aprotic solvent as a catalyst, with or without PVP in a molybdate method.
Morin and Prox in column 1 that the art recognizes that ferrous ammonium sulfate, stannous chloride and ascorbic acid have all been utilized in molybdate methods but are not considered satisfactory for several reasons. Stannous chloride is unstable, deviates from the Beer-Lambert law, has a sensitivity to change in acidity and has unstable color results, while FAS and ascorbic acid, while they obey the Beer-Lambert law, are slow acting and lack sensitivity. Slow reaction is stated to be at least 30 min.
It is concluded that the ideal molybdate system would have a reducing agent that is stable, acts in about 5 minutes, follows Beer's law, and produces stable color among its attributes.